application [106]. Further delineation of the 5 -flanking DNA of Emidentified a 50-bp DNA sequence that
is sufficient for ABA induction of GUS activity [107]. A DNA element, CACGTGGC, was conserved
among other ABA-induced genes, including rab16[108]. However, this element is also related to G-box
motifs, which are involved in light-induced gene expression [109]. This conserved element, which has been
referred to as an ABA-responsive element (ABRE), has been shown to bind nuclear proteins [83,108]. A
leucine zipper protein, EmBP-1, was identified and a cDNA cloned whose gene product binds the ABRE
ofEmin vitro [83]. This protein, EmBP-1, contains a leucine-zipper DNA binding motif, thought to be re-
sponsible for dimer formation, adjacent to a basic domain which is a cluster of positively charged amino
acids responsible for sequence-specific recognition. The combination of the basic domain and the leucine
zipper domain has been termed the bZIP domain. The basic domain of EmBP-1 is similar to that found in
other transcription factors that bind the DNA sequence element T/CACGTGGC, including TAF-1 from
tobacco, which binds the ABRE conserved in rab16genes [110], and GBF, which binds the G-box found
inrbcSgenes of tomato, Arabidopsis, and pea [109,111] (Figure 2). It is similar to other transcription fac-
tors that bind a similar DNA sequence element, TCCACGTAGA [112–114]. An element has been identi-
fied that confers ABA inducibility in a transient assay system, and a factor has been identified that can bind
this DNA element. However, the identification of several similar transcription factors that bind similar
DNA elements indicates that additional aspects of ABA inducibility are yet to be understood.
Additional specificity of the DNA elements may be derived from nucleotides surrounding the core
DNA sequence. Williams et al. [115] characterized the sequences flanking the G-box or ABRE core
CACGTG to determine how those DNA sequences affected binding. Based on the flanking sequence, the
G-box elements have been divided into two different classes to which bind two distinct classes of G-box
binding proteins. Further characterizations were made by using ACGT as the core and defining three dif-
ferent types of boxes by the nucleotide surrounding the core. EmBP-1, TAF-1, and HBP-1a were found
to have the greatest affinity for the CACGTGC sequence [116].
TAF-1 from tobacco [110] and EmBP-1 [83] from wheat both bind DNA elements with the core se-
quence CACGTGGC and have the same DNA-binding basic motif. However, neither motif I,
TACGTGGC [108], nor the hex tetramer, GGTGACGTGGC, can confer ABA responsiveness on a GUS
reporter gene in transgenic tobacco [117]. But a hex mutant, GGACGCGTGGC, with greatly reduced
affinity for TAF-1 can confer ABA responsiveness on a GUS reporter gene in transgenic tobacco [117].
These results indicate that there are multiple factors that can bind similar DNA motifs and that the exact
DNA sequence determines which factors will recognize it. In addition, although factors have similar
amino acid sequences within the DNA-binding domain, this information cannot be used to predict the ex-
act DNA elements these factors will bind in vivo. Other regions of the transcription factor besides the
DNA binding domain must also be important for determining DNA-binding specificity.
Since the studies on Em, other ABA-regulated genes have also been investigated. Another leagene,
rab28, is known to be regulated by endogenous ABA in maize [87]. This gene is expressed in vegetative
organs during periods of water deficit, and it has been demonstrated that an ABRE is involved in the reg-
ulation of rab28by ABA and water stress [118]. Chimeric genes, with a portion of the rab28 5
-flanking
DNA containing the ABRE and GUS, transfected into rice protoplasts were ABA responsive. An in vitro
dimethyl sulfate footprinting experiment identified guanine residues within the ABRE that are involved
in binding nuclear proteins [118]. Interestingly, when electrophoretic mobility shift assays were com-
pleted with proteins isolated from seeds or from drought-stressed leaves, complexes of two different sizes
were found. Both complexes were shown to bind the ABRE [118]. Therefore, it is proposed that the same
DNA sequence element or ABRE is involved in regulation of the expression of rab28in the seed during
development and in the leaf during stress, but the transcription factors and activators that are involved in
these two different types of regulation are not identical. Similarly, it has been shown that factors that bind
746 BRAYFigure 2 Basic domain motif of three bZIP proteins: EmBP-1 [83], TAF-1 [110], and O2 [112]. Identical
amino acids are shown in boldface type. The DNA-binding site for each of the bZIP is shown to the right of the
amino acid sequence.